The long-term goal of this proposal is to develop protocols for inducible and reversible tissue-specific gene silencing in the mouse. We propose to test the feasibility of an RNA interference (RNAi)- based strategy for gene inactivation in transgenic mice. RNAi is an experimental strategy that takes advantage of a conserved endogenous pathway for gene silencing. This pathway appears to serve a dual function: to guard cells against molecular parasites, and to regulate the chronology of development. RNAi is triggered by double-_stranded RNAs (dsRNA), corresponding to sense and antisense sequences of target genes. The dsRNA precursors are cleaved by Dicer RNAse into short interfering RNAs (siRNAs) that guide an RNA Induced Silencing Complex (RISC) to the target transcript. In a related mechanism, Dicer also cleaves dsRNA precursors to generate _short t emporal RNAs (stRNAs) that act by interfering with translation. RNAi is the most powerful technique available for the functional analysis of C.elegans and Drosophila genomes and systematic targeted inactivation projects are under way. The establishment of RNAi-based gene inactivation technologies in the mouse would have significant advantages over current protocols for gene inactivation. Because the gene silencing mechanism leaves the genomic locus intact, the silencing effect is potentially reversible. In addition, transgenic RNAi would be faster and significantly less costly than current locus inactivation protocols. Recent tissue culture studies provide strong evidence that the fundamental mechanisms for RNAi are evolutionarily conserved and operational in mammalian cells. However, the existence of the interferon response in mammalian cells is an obstacle to the implementation of dsRNA-based strategies. We intend to test four transgenic strategies in mice, each designed to avoid the activation of an interferon response. Our Specific Aims are: 1) To test whether gene silencing in transgenic mice can be accomplished by expression of short palindromic RNAs, which we term "snap-back" RNAs, 2) To test whether gene silencing can be accomplished by expression of long interrupted RNAs, which we term "bubble hybrid" RNAs, 3) To test whether gene silencing in the mouse can be facilitated by co-expression of Dicer RNAse and an RNAi transgene, and 4) To test whether "stRNA"-mediated inhibition of translation is feasible in transgenic mice. We will use two model systems to evaluate the efficacy of gene inactivation: the inhibition of tryrosinase expression in melanocytes and in the Retinal Pigmented Epithelium (RPE), predicting changes in pigmentation, and the inactivation of Rb in lens fiber cells resulting in cataracts and microphthalmia. Once established, RNAi technology will be more flexible than traditional gene knock-out techniques and will greatly accelerate the functional characterization of the mammalian genome.